DNA supercoiling affects almost all processes involving DNA in vivo. DNA topology affects cellular processes such as, for example, transcription, replication, packaging and segregation. Although the effects of DNA topology lead to significant alterations in cellular processes, much of the research devoted to understanding these processes is performed in vitro on substrates that do not adequately approximate physiological topology. For example, proteins that bind DNA (“DNA binding proteins”) often have different affinities and/or kinetics for DNA depending on the degree to which the DNA is supercoiled. Studying DNA binding by these proteins on substrates that do not approximate physiological topology is an inherent disadvantage with the DNA substrates used in these studies.
The use of DNA minicircles, for example, in binding studies has been suggested, but they have been difficult to produce and purify in significant quantity. DNA minicircles lack an origin of replication and are typically lost during cell division. However, if DNA minicircles are synthesized as linear molecules and then circularized by in vitro ligation, they do not exhibit physiological topology. When such minicircles pass through the linear stage, they lose their superhelicity. As only closed minicircles are topologically constrained, linear molecules must be ligated to form minicircles. However, yields are low and intermolecular ligation contaminants are prevalent when the short linear DNA molecules necessary for generating minicircles are used. Alternatively, without an origin of replication, such circles are not efficiently produced in large quantities in vivo.
Although there is promise for the use of minicircles for in vitro studies of DNA, e.g., for DNA binding assays, their use as a substrate is limited by their difficulty to be produce in large quantities while retaining a physiological topology.